Disregulation of the immune system is involved in numerous pathologies, and may be a factor that favours the establishment, maintenance or progression of disease. Deficient immune responses or immune suppression are known to enhance an animal's susceptibility to infection or to the development of cancer. Conversely, excessive or inappropriate immune responses are involved in the establishment or progression of unwanted inflammation or autoimmune conditions. It would thus be advantageous to be able to utilize agents that modulate immune responses, and to at least partially reverse-immune dysfunction when such dysfunction is a component of a given pathological condition.
The tumor suppressor protein p53 functions as a transcriptional factor that activates genes controlling cell cycle arrest and apoptosis (see, for example, Agarwal et al., 1998, J Biol Chem, 273(1): p1-4; Lakin & Jackson, 1999, Oncogene, 18(53): p7644-55; Sionov & Haupt, 1999, Oncogene, 18(45): p6145-6157). The activity of the p53 tumor suppressor protein and the c-Jun proto-oncogene are regulated by posttranslational modifications, such as phosphorylation or ubiquitination (Meek, 1999, Oncogene, 18(53): p7666-75). Specifically, covalent attachment of the ubiquitin-like modifier SUMO appears to modulate their transcriptional activity Rodriguez et al., 1999, Embo J, 18(22): p6455-61; Muller et al., 2000, J Biol Chem, 275(18): p13321-9).
Sumoylation proceeds via an enzymatic pathway that is mechanistically analogous to ubiquitination, but requires a different E1-activating enzyme and Ubc9, a SUMO-specific E2-conjugating enzyme (Lin et al., 2004, FEBS Lett, 573(1-3): p15-8). PIAS1 act as specific E3-like ligase that promotes sumoylation of p53 and c-Jun in vitro and in vivo. The PTAS proteins physically interact with both p53 and c-Jun and PIAS1 interacts with the tetramerization and C-terminal regulatory domains of p53 in yeast two-hybrid analyses (Megidish et al., 2002, J Biol Chem, 277(10): p8255-9). In addition, they bind to Ubc9, suggesting that they recruit the E2 enzyme to their respective substrate. The SUMO ligase activity requires the conserved zinc-finger domain, which is distantly related to the essential RING-finger motif, found in a subset of ubiquitin ligases.
PIAS proteins strongly repress the transcriptional activity of p53, suggesting that the PIAS-SUMO pathway plays a crucial role in the regulation of p53 and other transcription factors (Schmidt & Muller, 2002, Proc Natl Acad Sci USA, 99(5): p2872-7).
The STAT-1 transcription factor has been implicated as a tumor suppressor by virtue of its ability to inhibit cell growth and promotion of apoptosis. STAT-1 is required for optimal DNA damage-induced apoptosis. The basal level of the p53 inhibitor Mdm2 is increased in STAT-1(−/−) cells, suggesting that STAT-1 is a negative regulator of Mdm2 expression. STAT-1 interacts directly with p53, an association, which is enhanced following DNA damage. Therefore, in addition to negatively regulating Mdm2, STAT-1 also acts as a co-activator for p53. Hence STAT-1 is another member of a growing family of protein partners able to modulate the p53-activated apoptotic pathway (Townsend et al., 2004, J Biol Chem, 279(7): p5811-20).
Signal transducer and activator of transcription 1 (STAT1) mediates gene expression in response to cytokines and growth factors. Activation of STAT1 is achieved through its tyrosine phosphorylation, a process that involves Jak tyrosine kinases. One of these cytokines, IFN-gamma, induces STAT1 phosphorylation and leads to expression of multiple genes and apoptosis.
Viruses can evade the host immune system by inactivating different components of the IFN-activated JAK-STAT pathway. As described earlier, members of the Paramyxovirus family of RNA viruses target STATs for degradation. Epstein-Barr virus (EBV) inhibits the expression of IFN-receptor through the action of the EBV immediate-early protein, BZLF1 (Morrison et al., 2001, Immunity, 15(5): p787-99). Human cytomegalovirus inhibits IFN-induced expression of MHC class II molecules by selectively targeting JAK1 for degradation (Miller et al., 1998, J Exp Med, 187(5): p675-83). By contrast, infection with varicella-zoster virus inhibits the expression of STAT1 and JAK2, but not JAK1 (Abendroth et al., 2000, J Virol, 74(4): p1900-7). Individuals with defects in the IFN-JAK-STAT pathway show increased susceptibility to viruses and intracellular bacteria. Patients with mutations in the IFN-receptor chains are susceptible to infection with mycobacteria (Dupuis et al., 2000, Immunol Rev, 178: p129-37). Recently, patients with STAT1 deficiency have been reported (Dupuis et al., 2003, NatGenet, 33(3): p388-91). These individuals suffered from mycobacterial infection and died of lethal viral disease.
The aetiopathology of Crohn's disease—a chronic inflammatory bowel disease—is poorly understood. Mice with tissue-specific disruption of Stat3 during haematopoiesis show Crohn's disease-like pathogenesis (Welte et al., 2003, Proc Natl Acad Sci USA, 100(4): p1879-84). In addition, constitutively tyrosine phosphorylated STAT3 is found in intestinal T cells from patients with Crohn's disease (Lovato et al., 2003, J Biol Chem, 278(19): p16777-81). These results indicate that the dysregulation of STAT3 signaling might be involved in the pathogenesis of Crohn's disease. However, the exact role of STAT3 in the pathogenesis of Crohn's disease is not understood.
Apart from its affect on the JAK/STAT pathway, PIAS1 has been shown to be a negative regulator of the NF-KB signaling (Liu et al., 2005, Mol Cell Biol, 25(3): p1113-23). The NF-KB family of transcription factors is activated by a wide variety of signals to regulate a spectrum of cellular processes. The proper regulation of NF-KB activity is critical, since abnormal NF-KB signaling is associated with a number of human illnesses, such as chronic inflammatory diseases and cancer. Upon cytokine stimulation, the p65 subunit of NF-KB translocates into the nucleus, where it interacts with PIAS1. The binding of PIAS1 to p65 inhibits cytokine-induced NF-KB-dependent gene activation. PIAS1 blocks the DNA binding activity of p65 both in vitro and in vivo.
The ubiquitin-proteolysis system, which was discovered a little over 20 years ago by Hershko and Ciechanover, was originally thought to eliminate “old”, damaged, misfolded or misassembled proteins (Hershko & Ciechanover, 1998, Annu Rev Biochem, 67: p425-79; Hershko et al., 2000, Nat Med, 6(10): p1073-81). The system acquired its name from a 76-amino acid (aa) ubiquitously expressed protein, which is highly conserved in all eukaryotes. The ubiquitin pathway consists of several components that act sequentially in a hierarchical mode: a concerted two-step reaction that results in a high-energy thioester linkage between ubiquitin and a single conserved ubiquitin-activating enzyme (E1) and ubiquitin transfer through trans-acylation to one of several ubiquitin-conjugating enzymes (Ubcs or E2s). The latter collaborate with a large series of E3s (protein-ubiquitin ligases) in attaching ubiquitin molecules to the ε-amino group of the substrate's lysine residues, thus creating a reversible isopeptide bond. Pathways critical to cancer and immune regulation are regulated at several steps by polyubiquitination (Hershko & Ciechanover, 1998, supra; Ciechanover et al., 2000, J Cell Biochem Suppl, 34: p40-51; Schwartz & Hochstrasser, 2003, Trends Biochem Sci, 28(6): p321-8; Ben-Neriah, 2002, Nat Immunol, 3(1): p20-6).
Recent focus on the system has emphasized its role in controlling cellular processes via two modes of action. These are proteolysis-associated polyubiquitination for controlling the abundance of regulatory proteins and proteolysis-independent ubiquitination: mono-, multi- or polyubiquitination of regulatory proteins (Ciechanover et al., 2000, Bioessays, 22(5): p442-51). When the ubiquitins are linked to each other through the lysine amino acid found at position 48 of each ubiquitin, the target protein is directed to the cellular waste-disposal unit, the proteasome (Amit & Ben-Neriah, 2003, Semin Cancer Biol, 13(1):p15-28). If lysine 63 is used instead, it can serve as a signal for the target to assemble with other proteins (Wang et al., 2001, Nature, 412(6844): p346-51; Deng et al., 2000, Cell, 103(2): p351-61).
For proteolysis-associated ubiquitination, a further, poorly characterized, catalytic step is required: polymerization of a ubiquitin chain, which is facilitated by the same E2-E3 pair that attached the first ubiquitin molecule to the substrate or by additional enzymatic components. The polyubiquitin chain then serves as a recognition marker for the substrate-degrading 26S protein complex, the proteasome.
Parallel to the “classical” ubiquitination systems, there are other related enzymatic pathways that covalently attach ubiquitin-like molecules (Ubls) to target proteins for diverse purposes (Hochstrasser, 2000, Nat Cell Biol, 2(8): pE153-7; Jentsch & Pyrowolakis, 2000, Trends Cell Biol, 10(8): p335-42). Ubls are not only structurally related to ubiquitin, but conjugate to their protein targets through a ubiquitination-like enzymatic process, that is, formation of an isopeptide bond between the Ubl COOH-terminal glycine and an amino group of a target protein lysine. In addition, Ubl conjugation is done by enzymes that are related to ubiquitin pathway E1 and E2s (Hochstrasser, 2000, supra; Jentsch & Pyrowolakis, 2000, supra). Certain Ubl modifications may support protein ubiquitination: an example is the attachment of the Nedd8 Ubl to a subunit of the IB E3 protein that results in enhanced IB ubiquitination (Read et al., 2000, Mol Cell Biol, 20(7): p2326-33; Kawakami et al., 2001, Embo J, 20(15): p4003-12). Other Ubl modifications may interfere with protein ubiquitination, for example, the attachment of SUMO (small ubiquitin modifier) Ubl to IB, which suppresses its ubiquitination (Hay, 2001, Trends Biochem Sci, 26(5): p. 332-3), or have ubiquitination-unrelated functions, such as regulating nuclear protein export (Mahajan et al., 1997, Cell, 88(1): p97-107).
Whereas a single E1 activates ubiquitin, many (at least 25 in mammals) E2 species have been characterized in every eukaryotic organism. The multitude of E2 enzymes indicates that they specialize in distinct ubiquitination processes; however, the biochemical basis for this putative specialization is mostly unknown. Whereas E2 proteins are identified by their homology, the E3s constitute a highly heterogeneous class of proteins, which nevertheless can be classified into three groups: HECT (homologous to E6-AP COOH-terminus), RING and Ufd2-related (U-box) E3s (Weissman, 2001, Nat Rev Mol Cell Biol, 2(3): p169-78; Jackson et al., 2000, Trends Cell Biol, 10(10): p429-39). The HECT E3s are related to E6-associated protein (E6-AP)-the E3 that targets p53 in complex with papillomavirus E6 protein-and share a 350-aa HECT domain. HECT E3s have a unique mode of action: they catalyze ubiquitin transfer to the substrate through an intermediate thiol-ester between ubiquitin and a conserved cysteine in the HECT domain, In contrast, it appears that the RING E3s do not directly participate in the chemical transfer of ubiquitin to the substrate, but merely coordinate the activity of their associated E2s (Meroni & Diez-Roux, 2005, Bioessays, 27(11): p1147-57).
RING E3s are distinguished by the metal-coordinated RING-finger motif. The RING E3s are either single proteins with a substrate-targeting motif, such as an SH2 domain, or multi-subunit protein complexes in which substrate-targeting and the RING function are carried out by different proteins. Some of the most remarkable recent advances in the ubiquitin field have been made in characterizing the composition, partial structure and mode of substrate-recognition of three large multisubunit RING E3s: APC/C (anaphase-promoting complex-cyclosome), SCF (Skp1-cullin-1-F-box protein) and VCB (VHL-elongin C-elongin B complex) (Jackson et al., 2000, Trends Cell Biol, 10(10): p429-39; Deshaies et al., 1999, Annu Rev Cell Dev Biol, 15: p435-67; Zachariae & Nasmyth, 1999, Genes Dev, 13(16): p2039-58; Kondo & Kaelin, 2001, Exp Cell Res, 264(1): p117-25). U-box E3s constitute a newly identified class, some of which may mediate the assembly of polyubiquitin chains on proteins ubiquitinated by other E3s (Hatakeyama et al., 2001, J Biol Chem, 276(35): p33111-20).
Having a fundamental regulatory role in every eukaryotic organism, it is not surprising that proteolysis-associated ubiquitination also fulfills an important role in the immune system. Proteolysis-associated ubiquitination drives a variety of immunity-related regulatory events, from transcriptional activation to apoptosis (Shmueli & Oren, 2005, Cell, 121(7): p963-5). Parallel to well established proteolysis-associated ubiquitination, there are important proteosome-mediated degradation events in which the precise role of ubiquitination is still unclear; among the latter, antigen-processing is a prominent example (Kloetzel, 2001, Nat Rev Mol Cell Biol, 2(3): p179-87; Yewdell, 2001, Trends Cell Biol, 11(7): p294-7).
Ubiquitination of transcription factors can control their activity independently of proteosomal degradation. For example, Met4, a bZIP factor that regulates a large number of genes predominantly involved in methionine biosynthesis, is ubiquitinated but not degraded in the presence of high intracellular levels of S-adenosylmethionine (Kaiser et al., 2000, Cell, 102(3): p303-14). Ubiquitination inactivates Met4 at least in part because it precludes recruitment of the coactivator, Cbfl (Kaiser et al., 2000, supra); in addition, binding of Met4 to a subclass of its target promoters is compromised by ubiquitination (Kuras et al., 2002, Mol Cell, 10(1): p69-80). Ubiquitination does not necessarily inhibit transcription factors since ubiquitination of the HIVTat protein by Mdm2 augments its ability to activate transcription (Bres et al., 2003, Nat Cell Biol, 5(8): p754-61). Similarly, ubiquitination of Myc by Skp2 contributes to transcriptional activation, potentially by allowing Myc to recruit proteasomal subunits that have a proteolysis-independent role in transcriptional activation (Ferdous et al., 2001, Mol Cell, 7(5): p981-91). Two signals are known to determine whether ubiquitination leads to degradation. Proteolytic substrates are modified by polyubiquitin chains, and a minimum chain length of about four ubiquitin residues appears to be required to target the attached protein to the proteasome (Flick et al., 2004, Nat Cell Biol, 6(7): p634-41). The lysine residue of ubiquitin, used for polyubiquitin chain formation, specifies the second signal. Whereas chains linked through lysine 48 usually lead to proteasomal degradation, those linked through lysine-63 of ubiquitin do not target proteins to the proteasome (Bres et al., 2003, supra).
The TRIM/RBCC proteins are defined by the presence of the tripartite motif composed of a RING domain, one or two B-box motifs and a coiled-coil region (Reymond et al., 2001, Embo J, 20(9): p2140-51). These proteins are involved in a plethora of cellular processes such as apoptosis, cell cycle regulation and viral response. Consistently, their alteration results in many diverse pathological conditions. The highly conserved structure of these proteins suggests that a common biochemical function may underlie their assorted cellular roles. Some TRIM/RBCC proteins are implicated in ubiquitination and propose that this large protein family represents a novel class of ‘single protein RING finger’ ubiquitin E3 ligases (Meroni & Diez-Roux, 2005, supra).
Ubiquitin ligases play a key role in protein localization, transcriptional modulation and protein turnover within the cell. Modulation of these targets presents a novel approach to treating diseases where the normal cell processes are out of balance, such as in cancer where the cell cycling is abnormal. Ubiquitin ligase cancer targets play a role in the regulation of stability, localization, and activity of key proteins such as oncoproteins and tumour suppressor genes. Ubiquitin ligase targets are numerous and modular. This provides the potential for intervening in a highly specific fashion in a disease, potentially improving efficacy and minimizing side-effects.
It can be seen that transcription factors play a major role in homeostasis, especially with respect to the immune system. Accordingly, if modulators or regulators of transcription factors like those discussed above can be identified it might be possible to regulate cell proliferation, migration, and/or differentiation.